In the first cycle following transfer from a 12 h light-12 h dark cycle (LD12:12) to constant darkness (DD), the standard deviation in circadian phase among individual clocks in populations of Gonyaulax polyedra is approximately 60 min. When a culture is transferred to constant light conditions (LL) from an LD 12:12 cycle, the standard deviation increases in the first 2-3 d, but then remains unchanged, suggesting a lack of observable desynchronization in LL after the transient period. The synchrony in a cell population is preserved even after several cell divisions. The results indicate that variations in period among cells are small, that the period of an individual clock does not fluctuate randomly from day to day, and that the circadian phase of a mother cell is faithfully passed to the clocks of the daughter cells.

Animals↗

PubMedDate not supplied

A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies.

Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state or time-resolved fluorescence resonance energy transfer (FRET) detection of the kinetics of protein folding require relatively large quantities (approximately 10-100 mg) of site-specific doubly labeled protein samples. Engineered cysteine residues are common targets for labeling of proteins. The challenge here is to develop methods for selective modification of one of two reactive sulfhydryl groups in a protein molecule. A general systematic procedure for selective labeling of each of two cysteine residues in a protein molecule was developed, using Escherichia coli adenylate kinase (AKe) as a model protein. Potential sites for insertion of cysteine residues were selected by examination of the crystal structure of the protein. A series of single-cysteine mutants was prepared, and the rates of the reaction of each engineered cysteine residue with a reference reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] were determined. Two-cysteine mutants were prepared by selection of pairs of sites for which the ratio of this reaction rate constant was high (>80). The conditions for the selective labeling reaction were optimized. In a first cycle of labeling, the more reactive cysteine residue was labeled with a fluorescent probe (donor). The second probe was attached to the less reactive site under unfolding conditions in the second cycle of labeling. The doubly and singly labeled mutants retained full enzymatic activity and the capacity for a reversible folding-unfolding transition. High yields (70-90%) of the preparation of the pure, site-specific doubly labeled AK mutant were obtained. The procedure described herein is a general outline of procedures, which can meet the double challenge of both site specificity and large-scale preparation of doubly labeled proteins.

Adenylate Kinase↗

PubMedDate not supplied

Design consideration and probes for fluorescence resonance energy transfer studies.

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 A in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.

Acetamides↗

PubMedDate not supplied

Micromethod for the assay of renin of seven species.

Renin was extracted and purified from human, dog, hog, rat, beef, rabbit and sheep kidneys. The renin "concentration" of these preparations was determined and expressed in international (Goldblatt) units by measuring the pressor effect produced by intravascular injection into normal dogs of a permanent colony. The renin "activity" was determined by bioassay, in the rat, of the angiotensin produced by incubation in vitro with renin substrate prepared from the serum of nephrectomized dogs. The rate of angiotensin formation was proportional to the concentration of renin, independent of the substrate concentration, and rather similar for renin of all seven species (mean rate = 55 x 10(4) ng angiotensin/unit renin/16 hrs). Due to this uniformity, either of the two international reference preparations of renin (human or hog) from the World Health Organization can serve as an internal standard in the assay of reninof each of the seven species, to express their concentration in terms of the international unit. Renin substrate from hog plasma was suitable for the assay of human, dog and hog renin (mean rate = 55 x 10(4)). However, it reacted much more slowly with the renin of rat, beef, rabbit and sheep (mean rate = 9 x 10(4)) and was, therefore, less suitable for their assay.

Angiotensins↗